In vitro techniques provide a well controlled environment for the study of cell differentiation. The odontoblasts are the cells responsible for dentin formation during normal development and during repair. The factors which induce the differentiation of these cells are not well understood. The long term goal is to elucidate the mechanism of odontoblast differentiation and identify factors that stimulate this process. These can then be used for clinical application for promotion of dentin repair. Dental papilla cells were removed from newborn rat molars and cultured on Type I collagen coated dishes containing DMEM supplemented with 10% FBS and antibiotics. Addition of ascorbic acid and dexamethasone resulted in the appearance of mineralized areas. Morphological observations suggested that dental papilla cells may differentiate into odontoblasts under these conditions. Immunolocalization with anti rat dentin siaoloprotein antibody showed that this antibody to a dentin marker protein binds the mineralized matrix produced by these cells. Immunoprecipitation of phosphoproteins purified from conditioned media demonstrates a protein that migrates on SDS polyacrylamide electrophoresis with an apparrent molecular weight consistent with rat dentin sialoprotein. The establishment of an in vitro system for examining odontablasts will allow the study of the effects of putative pulp treatment agents on normal odontoblast differentiation and dentin formation. Keys words: odontoblast, dental papilla, tissue culture, immunocytechemistry, electron microscopy